In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5β-androstane 3α,17β-diol and <em>5α</em>-androstane 3α,17β-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (<em>DHT</em>) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.

OBJECTIVE

While an association between androgens and different types of aggression has been well documented in male offenders, the influence of androgens on externalizing behavior in adolescents at risk for antisocial behavior has not been investigated so far.

METHODS

Plasma levels of the main androgen metabolites testosterone (T) and <em>5a</em>-dihydrotestosterone (<em>DHT</em>) were measured in N = 119 14-year-olds (51 boys, 68 girls) from a prospective longitudinal study of children at risk. The Achenbach Child Behavior Checklist (CBCL) and the Youth Self Report Form (YSR) were used to assess externalizing behavior at age 14.

RESULTS

The CBCL revealed significant positive correlations between DHT levels and the subscales "externalizing problems" and the problem scales "aggressive behavior" and "delinquent behavior" in male adolescents. Only the YSR subscale "delinquent behavior" exhibited a marginally significant association with DHT. Neither scale showed any significant correlations between androgen levels and externalizing behavior in female adolescents.

CONCLUSIONS

Earlier findings of androgen effects on aggressive and antisocial behavior in male offenders were confirmed for male adolescents from a general population sample. The results stress the importance of the androgen metabolite DHT.

Finasteride and Dutasteride are <em>5α</em>-reductase inhibitors used in the clinic to treat endocrine conditions and were recently found to modulate brain dopamine (DA) neurotransmission and motor behavior. We investigated if Finasteride and Dutasteride have a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) male mice as a model of Parkinson's disease (PD). Experimental groups included saline treated controls and mice treated with saline, Finasteride (5 and 12.5 mg/kg) or Dutasteride (5 and 12.5 mg/kg) for 5 days before and 5 days after MPTP administration (4 MPTP injections, 6.5 mg/kg on day 5 inducing a moderate DA depletion) and then they were euthanized. MPTP administration decreased striatal DA contents measured by HPLC while serotonin contents remained unchanged. MPTP mice treated with Dutasteride 5 and 12.5 mg/kg had higher striatal DA and metabolites (DOPAC and HVA) contents with a decrease of metabolites/DA ratios compared to saline-treated MPTP mice. Finasteride had no protective effect on striatal DA contents. Tyrosine hydroxylase (TH) mRNA levels measured by in situ hybridization in the substantia nigra pars compacta were unchanged. Dutasteride at 12.5 mg/kg reduced the effect of MPTP on specific binding to striatal DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) measured by autoradiography. MPTP reduced compared to controls plasma testosterone (T) and dihydrotestosterone (<em>DHT</em>) concentrations measured by liquid chromatography-tandem mass spectrometry; Dutasteride and Finasteride increased plasma T levels while <em>DHT</em> levels remained low. In summary, our results showed that a <em>5α</em>-reductase inhibitor, Dutasteride has neuroprotective activity preventing in male mice the MPTP-induced loss of several dopaminergic markers.

<em>5α</em>-reductase (<em>5α</em>-R) deficiency is an important cause of ambiguious genitalia in genetic males; however therapeutic experience in literature is limited. In this report authors describe a child with 46 XY Disorder of Sexual Differentiation (DSD), due to <em>5α</em>-reductase deficiency, who was managed with Dihydrotestosterone (<em>DHT</em>) gel.

Spermatogenesis and sperm maturation are androgen-dependent processes. Steroid <em>5α</em>-reductase (SRD5A) is a key enzyme converting testosterone into the more active metabolite, dihydrotestosterone (<em>DHT</em>). We aimed to investigate the association between the genetic variants of SRD5A2 (rs4952197, rs2268797, rs13395648, rs523349 and rs632148) and semen quality. Variant genotyping and semen analysis was performed in 708 males with definite idiopathic infertility by TaqMan SNP genotyping assays and computer-assisted semen analysis, respectively. It was found that the rs13395648 TC genotype was associated with a significantly lower semen volume compared with the TT genotype (P=0.016). The same trend was found between the combination of the TC and CC genotypes and the TT genotype (P=0.020). With regard to variant rs632148, subjects with the GC genotype had significantly lower sperm motility in comparison to those with the GG genotype (P=0.029). The sperm motility between the combination of the GC and CC genotypes and the GC genotype was also significantly different (P=0.033). These findings indicate that genetic variants in the SRD5A2 gene may be associated with semen quality.

Recent advances in stem cell biology have resulted in identifying new agents to differentiate stem cell-derived-neural cells. Different stem cell types have been shown to differentiate into neural cells. It has been shown that P19 line of embryonal carcinoma cells develops into neurons and astroglia after exposure to some hormones such as dehydroepiandrosterone (DHEA). Steroid <em>5α</em>-reductase is a key enzyme in the conversion of several Δ4-3 keto steroids, such as testosterone into their respective <em>5α</em>-reductase derivatives. Finasteride is a <em>5α</em>-reductase inhibitor that inhibits conversion of testosterone to the more potent androgen dihydrotestosterone (<em>DHT</em>). Reduction in <em>DHT</em> and sustaining testosterone levels has an important impact on differentiation and proliferation of embryonal carcinoma cells to neural cells. We hypothesize that finasteride, a <em>5α</em>-reductase inhibitor, will be differentiate embryonal carcinoma cell to the neural cell and increase their proliferation due to the elevation levels of testosterone, a neuroprotective neurosteroid.

OBJECTIVE

Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue.

METHODS

Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand <em>5α</em>-dihydrotestosterone (<em>DHT</em>), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry.

RESULTS

DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels.

CONCLUSIONS

In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

Eplerenone (Inspra) is an aldosterone receptor antagonist approved for the treatment of hypertension and heart failure after a myocardial infarction. In vitro receptor binding and transactivation studies showed eplerenone had high selectivity for the mineralocorticoid receptor over other steroid receptors (glucocorticoid, androgen, and progesterone). The most sensitive off-target effect of orally administered eplerenone preclinically was prostate atrophy in dogs. Dose-related prostate atrophy was observed at eplerenone dosages ≥15 mg/kg/day for 13 weeks or longer. The no observed adverse effect level (NOAEL) for the prostate effect in dogs was 5 mg/kg/day. The maximal effect was seen by 13 weeks and the atrophy was reversible even after 1 year of daily treatment. An additional study demonstrated dogs with eplerenone-induced prostate atrophy (confirmed by intrarectal ultrasound) had slightly decreased semen volume but no compound-related effects on libido, semen protein content, sperm motility, daily sperm production, or epididymal sperm transit time. Four possible mechanisms for prostate effect were investigated: (1) inhibition of testosterone synthesis and secretion; (2) inhibition of <em>5α</em>-reductase, the enzyme within the prostate that converts testosterone into the more active growth factor dihydrotestosterone (<em>DHT</em>); (3) competitive antagonism of the androgen receptor; and (4) inhibition of <em>5α</em>-reductase or competitive antagonism of the androgen receptor by aldosterone, which increased in dogs treated with eplerenone. Data from these studies supported blockade of androgen receptors at suprapharmacological concentrations of eplerenone. Another mineralocorticoid blocker, spironolactone, had greater antiandrogenic activity than eplerenone both in vivo and in vitro, and it has well known clinically significant antiandrogenic effects in humans, whereas eplerenone does not.

The steroid hormone <em>5α</em>-dihydrotestosterone (<em>DHT</em>) is one of the most physiologically important androgens in male vertebrates, with the exception of teleost fish, in which it is generally assumed that <em>DHT</em> does not play any major physiological role. However, this assumption is challenged by the fact that all the components involved in <em>DHT</em> biosynthesis and action are present and evolutionary conserved in teleost fish. In fact, testosterone (T) is converted into <em>DHT</em> by two isoforms of the enzyme steroid-5-alpha-reductase (<em>5α</em>R), and both <em>5α</em>Rs gene expression and enzymatic activity have been detected in several tissues of different teleost species, which also have an androgen receptor with high binding affinity to <em>DHT</em>. This body of evidence strongly suggest that <em>DHT</em> is synthesised by teleost fish. We investigated this hypothesis using the cyprinid fathead minnow (Pimephales promelas) as the experimental model. The study of the evolutionary and functional conservation of <em>5α</em>Rs in teleost fish was used to support the experimental approach, based on an ultrasensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method to identify and measure simultaneously T and <em>DHT</em> in fathead minnow biological fluids and tissues. The analyses were performed using plasma samples collected from both male and female adult fish and samples of testicular tissue collected from sexually mature males. Both T and <em>DHT</em> were identified and quantified in all the samples analysed, and in particular, the high concentrations of <em>DHT</em> quantified in the testes suggested that these organs are a likely site of synthesis of <em>DHT</em> in the teleost fathead minnow, as they are in mammals. These results may represent the basis for future studies aimed at elucidating the physiological role, if any, of <em>DHT</em> in teleost fish.

Androgens have been associated with the development of normal breast, and their role in mammary gland carcinogenesis has also been described. Several studies reported that androgens inhibit breast cancer cell growth, whereas others linked their action with the modulation of calcium (Ca(2+)) pumps, Ca(2+) channels and Ca(2+)-binding proteins. Also, it is known that deregulated Ca(2+) homeostasis has been implicated in the pathophysiology of breast. The L-type Ca(2+) channels (LTCCs) were found to be up-regulated in colon, colorectal and prostate cancer, but their presence in breast tissues remains uncharacterized. On the other hand, regucalcin (RGN) is a Ca(2+)-binding protein involved in the control of mammary gland cell proliferation, which has been identified as an androgen target gene in distinct tissues except breast. This study aimed to confirm the expression and activity of LTCCs in human breast cancer cells and investigate the effect of androgens in regulating the expression of α1C subunit (Cav1.2) of LTCCs and Ca(2+)-binding protein RGN. PCR, Western blot, immunofluorescence and electrophysiological experiments demonstrated the expression and activity of Cav1.2 subunit in MCF-7 cells. The MCF-7 cells were treated with 1, 10 or 100 nM of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) for 24-72 h. The obtained results showed that 1 nM <em>DHT</em> up-regulated the expression of Cav1.2 subunit while diminishing RGN protein levels, which was underpinned by reduced cell viability. These findings first confirmed the presence of LTCCs in breast cancer cells and opened new perspectives for the development of therapeutic approaches targeting Ca(2+) signaling.

The aim of this study was to search for clinical and laboratorial data in 46,XY patients with ambiguous genitalia (AG) and normal testosterone (T) synthesis that could help to distinguish partial androgen insensitivity syndrome (PAIS) from <em>5α</em>-reductase type 2 deficiency (<em>5α</em>-RD2) and from cases without molecular defects in the AR and SRD5A2 genes. Fifty-eight patients (51 families) were included. Age at first evaluation, weight and height at birth, consanguinity, familial recurrence, severity of AG, penile length, LH, FSH, T, dihydrotestosterone (<em>DHT</em>), Δ4-androstenedione (Δ4), and T/<em>DHT</em> and T/Δ4 ratios were evaluated. The AR and SRD5A2 genes were sequenced in all cases. There were 9 cases (7 families) of <em>5α</em>-RD2, 10 cases (5 families) of PAIS, and 39 patients had normal molecular analysis of SRD5A2 and AR genes. Age at first evaluation, birth weight and height, and T/<em>DHT</em> ratio were lower in the undetermined group, while penile length was higher in this group. Consanguinity was more frequent and severity of AG was higher in <em>5α</em>-RD2 patients. Familial recurrence was more frequent in PAIS patients. Birth weight and height, consanguinity, familial recurrence, severity of AG, penile length, and T/<em>DHT</em> ratio may help the investigation of 46,XY patients with AG and normal T synthesis.

The enzyme 5alpha-reductase 1 (<em>5α</em>-R(1)) that converts testosterone (T) to dihydrotestosterone (<em>DHT</em>) is present in many mammalian tissues including the spinal cord. It is established that morphine administration decreases spinal cord T levels, but the mechanism is still undetermined. Here, we investigated the link between T and the enzyme <em>5α</em>-R(1) in the spinal cord after morphine administration. For spinal cord steroid extraction, all the animals were killed 30 min, 2 h (acute) and 14 days (chronic) after first drug injection by decapitation. The whole spinal cord was removed and kept frozen at -20°C until T and <em>DHT</em> extraction. The effects of acute and chronic morphine administration on <em>5α</em>-R(1) expression in the adult male rat spinal cord were evaluated using RT-PCR. Spinal cord T and <em>DHT</em> levels were measured using radioimmunoassay before and after the morphine exposure. Morphine significantly reduced the T concentration after acute and chronic exposure in the spinal cord. In contrast, the <em>5α</em>-R(1) expression and of course <em>DHT</em> levels increased the following chronic morphine administration. One important reason for the decreasing effect of morphine exposure on the spinal cord T level is due to an increase in the <em>5α</em>-R(1) levels. We suggest that morphine plays a regulatory role in metabolism of neurosteroids, especially T in the spinal cord via <em>5α</em>-R(1).

Master N had genital malformation at birth and had bilateral gonads in the labial fold. He was reared as a boy and corrective surgery was done at the age of 4 years and was reassessed at the age of 14 years. His testosterone/dihydrotestosterone (<em>DHT</em>) was 11.8 (reference range <=10). Molecular analysis of SRD5A2 gene indicated the presence of a novel heterozygous missense mutation of p.A52T in exon 1, which was also detected in mother. The father, sister and maternal grandfather were found to have normal SRD5A2 gene sequence. We also detected an intronic (1-2) homozygous T>C transition in patient, whereas both parents were found to have the same transition in heterozygous form. Although <em>5α</em>-steroid reductase 2 deficiency is an autosomal-recessive disorder, in this case, it appears that there may be a dominant inheritance because only one identified mutation was present which was passed from mother to son.

Clinical records (n = 24) with an established diagnosis of <em>5α</em>-reductase-2 deficiency were reviewed. A previous misdiagnosis was present in about 70% (period from first observation to definitive diagnosis: 9.1 ± 10.8 years), and in 8 children gonadal removal was performed before certain diagnosis. Initial sex assignment was female in 16/24 (67%) and male in 8/24 (33%) cases. After diagnosis, sex re-assignment was performed in 5 babies (4 girls to male sex; 1 boy to female sex). Baseline testosterone/<em>DHT</em> ratio was diagnostic in 6/12 subjects (first months of life n = 4; puberty n = 2), while post-hCG testosterone/<em>DHT</em> ratio was diagnostic in all tested individuals (choosing both the cut-off value 15 or 10). Eighteen different mutations in the steroid-<em>5α</em>-reductase-2 (SRD5A2) gene were identified, 5 of which have never been reported. In conclusion, a time lag exists before the diagnosis of <em>5α</em>-reductase-2 deficiency is established; sex assignment and gonadal removal may be performed before certain diagnosis. Sex re-assignment is usually female to male, but the contrary may occur. A large variability in clinical phenotypes and genetic mutations was present in this cohort. Accurate endocrine evaluation is recommended in babies possibly affected by <em>5α</em>-reductase-2 deficiency, since the use of appropriate cut-off values of testosterone/<em>DHT</em> ratio after hCG stimulation may permit to select individuals for SRD5A2 gene analysis. A genotype-phenotype correlation was not found in this study.

Background: Low-grade chronic inflammation is commonly seen in polycystic ovary syndrome (PCOS) patients with elevated levels of inflammatory cytokines in the endometrium. However, our understanding of the mechanisms underlying cytokine synthesis and increased endometrial inflammation in PCOS patients remains limited.

Methods: Endometrial biopsy samples were collected from non-PCOS (n = 17) and PCOS (n = 22) patients either during the proliferative phase of the menstrual cycle or with hyperplasia. Endometrial explants were prepared from PCOS patients and subjected to pharmacological manipulation in vitro. The expression and localization of TLR2/4, key elements of innate immune signal transduction and NFκB signaling pathways, and multiple cytokines were comprehensively evaluated by Western blotting, immunohistochemistry, and immunofluorescence in endometrial tissues.

<strong class="sub-title"> Results: </strong> We demonstrated the distribution of protein expression and localization associated with the significantly increased androgen receptor, TLR2, and TLR4-mediated activation of IRF-7 and NFkB signaling, cytokine production, and endometrial inflammation in PCOS patients compared to non-PCOS patients with and without endometrial hyperplasia. In vitro experiments showed that <em>5α</em>-dihydrotestosterone (<em>DHT</em>) enhanced androgen receptor, TLR4, IRF-7, and p-NFκB p65 protein expression along with increased IFNα and IFNɣ abundance. The effects of <em>DHT</em> on IRF-7, p-NFκB p65, and IFN abundance were abolished by flutamide, an anti-androgen. Although 17β-estradiol (E2) decreased p-IRF-7 expression with little effect on TLR-mediated IRF7 and NFκB signaling or on cytokine protein levels, exposure to metformin alone or in combination with E2 suppressed IRAK4, p-IRF-7, IRF-7, IKKα, p-NFκB p65, IFNɣ, and TNFα protein expression.

Conclusion: Cytokine synthesis and increased endometrial inflammation in PCOS patients is coupled to androgen-induced TLR4/IRF-7/NFkB signaling, which is be inhibited by metformin treatment.

Keywords: TLR4/IRF7/NFκB; androgen; endometrial inflammation; innate immune response; polycystic ovary syndrome.

Androgens act in concert with vitamin D to influence reabsorption of calcium. However, it is unclear whether androgens directly regulate vitamin D homeostasis or control other cellular events that are related to vitamin D metabolism. To examine whether the expression of vitamin D-related genes in mouse kidney is driven by androgens or androgen-dependent effects, the androgen receptor and other sex steroid receptors were monitored in orchidectomized mice treated with <em>5α</em>-dihydrotestosterone (<em>DHT</em>). Our results revealed that exposing orchidectomized mice to <em>DHT</em> inhibited the expression of progesterone receptor (Pgr) with or without estrogen receptor α expression, the latter was confirmed by ER-positive (MCF7 and T47D) or -negative (PCT) cells analysis. The loss of Pgr in turn decreased the expression of renal 24-hydroxylase via transcriptional regulation because Cyp24a1 gene has a progesterone receptor-binding site on promoter. When male kidneys preferentially hydroxylate 25-hydroxyvitamin D3 using 24-hydroxylase rather than 25-hydroxyvitamin D3-1-alpha hydroxylase, <em>DHT</em> suppressed the Pgr-mediated 24-hydroxylase expression, and it is important to note that <em>DHT</em> increased the blood 25-hydroxyvitamin D3 levels. These findings uncover an important link between androgens and vitamin D homeostasis and suggest that therapeutic modulation of Pgr may be used to treat vitamin D deficiency and related disorders.

The effects of some novel steroidal compounds were evaluated against both human C17,20-lyase and <em>5a</em>lpha-reductase in vitro and also against androgen synthesis in normal male rats. L-2, L-36, L-37, and I-41 showed potent inhibition of human testicular C17,20-lyase, with IC50s of 43, 39, 42, and 58 nM, respectively. In contrast, ketoconazole, a competitive inhibitor of C17,20-lyase, had an IC50 of 76 n.M. L-36 also showed potent inhibitory activity against <em>5a</em>-reductase in human prostatic microsomes, with an IC50 of approximately 31 nM. The inhibitory activities of L-2 and 1-41 on <em>5a</em>lpha-reductase were moderate, with IC50s of 75 and 151 nM, respectively, whereas L-37 showed little inhibitory activity against this enzyme. In comparison, finasteride, a potent inhibitor of <em>5a</em>lpha-reductase, had an IC50 of 33 nM. When normal male rats were treated with these novel compounds (50 or 100 mg/kg/day) for 14 consecutive days, the wet weight of the prostate was significantly reduced by L-36, L-37, and I-41, compared to the control group. Testosterone levels in rat serum were also reduced by L-36 (55%), L-37 (86%), and I-41 (53%). The concentrations of testosterone in rat testes were reduced by these novel compounds by 13-74%. The compounds also reduced the concentration of testosterone in rat prostates by 35-75%. Similarly, dihydrotestosterone (<em>DHT</em>) concentration in rat serum was reduced 30-89% by these compounds, compared to the control group. Prostatic <em>DHT</em> levels were also lower in rats treated with L-36 (48%), L-37 (54%), or I-41 (26%). In contrast, L-2 enhanced serum testosterone and prostatic <em>DHT</em> concentrations by >50%. These findings suggest that the dual activities of several of these novel inhibitors of C17,20-lyase and <em>5a</em>lpha-reductase accounts for the diminished levels of circulating androgens in vivo.

Purpose: A major mechanism of castration-resistant prostate cancer (CRPC) involves intratumoral biosynthesis of dihydrotestosterone (<em>DHT</em>) from adrenal precursors. We have previously shown that adrenal-derived androstenedione (AD) is the preferred substrate over testosterone (T) for <em>5α</em>-reductase expressed in metastatic CRPC, bypassing T as an obligate precursor to <em>DHT</em>. However, the metabolic pathway of adrenal-derived <em>DHT</em> biosynthesis has not been rigorously investigated in the setting of primary disease in the prostate.Experimental Design: Seventeen patients with clinically localized prostate cancer were consented for fresh tissues after radical prostatectomy. Prostate tissues were cultured ex vivo in media spiked with an equimolar mixture of AD and T, and stable isotopic tracing was employed to simultaneously follow the enzymatic conversion of both precursor steroids into nascent metabolites, detected by liquid chromatography-tandem mass spectrometry. CRPC cell line models and xenograft tissues were similarly assayed for comparative analysis. A tritium-labeled steroid radiotracing approach was used to validate our findings.Results: Prostatectomy tissues readily <em>5α</em>-reduced both T and AD. Furthermore, <em>5α</em>-reduction of AD was the major directionality of metabolic flux to <em>DHT</em>. However, AD and T were comparably metabolized by <em>5α</em>-reductase in primary prostate tissues, contrasting the preference exhibited by CRPC in which AD was favored over T. <em>5α</em>-reductase inhibitors effectively blocked the conversion of AD to <em>DHT</em>.Conclusions: Both AD and T are substrates of <em>5α</em>-reductase in prostatectomy tissues, resulting in two distinctly nonredundant metabolic pathways to <em>DHT</em>. Furthermore, the transition to CRPC may coincide with a metabolic switch toward AD as the favored substrate. Clin Cancer Res; 23(20); 6351-62. ©2017 AACR.

Estrogenic and androgenic steroids synthesized in the brain may rapidly modulate synaptic plasticity interacting with specific membrane receptors. We explored by electrophysiological recordings in hippocampal slices of male rat the influence of 17β-estradiol (E2) and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) neo-synthesis on the synaptic changes induced in the CA1 region. Induction of long-term depression (LTD) and depotentiation (DP) by low frequency stimulation (LFS, 15 min-1 Hz) and of long-term potentiation (LTP) by high frequency stimulation (HFS, 1 s-100 Hz), medium (MFS, 1 s-50 Hz), or weak (WFS, 1 s-25 Hz) frequency stimulation was assayed under inhibitors of enzymes converting testosterone (T) into <em>DHT</em> (<em>5α</em>-reductase) and T into E2 (P450-aromatase). We found that LFS-LTD depends on <em>DHT</em> synthesis, since it was fully prevented under finasteride, an inhibitor of <em>DHT</em> synthesis, and rescued by exogenous <em>DHT</em>, while the E2 synthesis was not involved. Conversely, the full development of HFS-LTP requires the synthesis of E2, as demonstrated by the LTP reduction observed under letrozole, an inhibitor of E2 synthesis, and its full rescue by exogenous E2. For intermediate stimulation protocols <em>DHT</em>, but not E2 synthesis, was involved in the production of a small LTP induced by WFS, while the E2 synthesis was required for the MFS-dependent LTP. Under the combined block of <em>DHT</em> and E2 synthesis all stimulation frequencies induced partial LTP. Overall, these results indicate that <em>DHT</em> is required for converting the partial LTP into LTD whereas E2 is needed for the full expression of LTP, evidencing a key role of the neo-synthesis of sex neurosteroids in determining the direction of synaptic long-term effects.

The impact of testosterone (T) treatment on antidoping detection tests in female-to-male (F2M) transgender men is unknown. We investigated urine and serum sex steroid and luteinizing hormone (LH) profiles in T-treated F2M men to determine whether and, if so, how they differed from hypogonadal and healthy control men.

Healthy transgender (n = 23) and hypogonadal (n = 24) men aged 18 to 50 years treated with 1000 mg injectable T undecanoate provided trough urine and blood samples and an additional earlier postinjection sample (n = 21). Healthy control men (n = 20) provided a single blood and urine sample. Steroids were measured by mass spectrometry-based methods in urine and serum, LH by immunoassay, and uridine 5'-diphospho-glucuronosyltransferase 2B17 genotype by polymerase chain reaction.

Urine LH, human chorionic gonadotropin, T, epitestosterone (EpiT), androsterone (A), etiocholanolone (Etio), A/Etio ratio, dehydroepiandrosterone (DHEA), dihydrotestosterone (<em>DHT</em>), and <em>5α</em>,3α- and 5β,3α-androstanediols did not differ between groups or by time since last T injection. Urine T/EpiT ratio was <4 in all controls and 12/68 (18%) samples from T-treated men, but there was no difference between T-treated groups. Serum estradiol, estrone, and DHEA were higher in transgender men, and serum T and <em>DHT</em> were higher in earlier compared with trough blood samples, but serum LH, follicle-stimulating hormone, and 3α- and 3β,<em>5α</em>-diols did not differ between groups.

Urine antidoping detection tests in T-treated transgender men can be interpreted like those of T-treated hypogonadal men and are unaffected by time since last T dose. Serum steroids are more sensitive to detect exogenous T administration early but not later after the last T dose.

Endocrine disrupting chemicals (EDCs) are common pollutants in the environment and can induce disruption of the endocrine and immune systems. The present study evaluated the effects of selected common environmental EDCs on secretion of inflammatory biomarkers by RAW264.7 cells. The EDCs investigated were Estradiol (E2), <em>5α</em>-dihydrotestosterone (<em>DHT</em>), and Bisphenol A (BPA). To evaluate if the effects caused by EDCs were modulated by steroid hormone receptors, antagonists of estrogen and androgen receptors were used. The steroid receptor antagonists used were Tamoxifen, an estrogen receptor antagonist, and Flutamide, an androgen receptor antagonist. Secretion of biomarkers of inflammation, namely nitric oxide (NO) and interleukin 6 (IL-6), were monitored. The NO was determined using Griess reaction and IL-6 was measured by enzyme linked immunosorbent assay (ELISA). Although 5 μg/mL E2, <em>DHT</em>, and BPA were not toxic to RAW264.7 cell cultures, the same treatments significantly (<i>p</i> < 0.001) reduced both NO and IL-6 secretion by lipopolysaccharide (LPS)-stimulated RAW264.7 cell cultures. The suppression of NO and IL-6 secretion indicate inhibition of inflammation by <em>DHT</em>, E2, and BPA. The inhibitory effects of <em>DHT</em>, E2 and BPA are partially mediated via their cellular receptors, because the effects were reversed by their respective receptor antagonists. Flutamide reversed the effects of <em>DHT</em>, while Tamoxifen reversed the effects of E2 and BPA. In conclusion, E2, BPA, and <em>DHT</em> inhibit the synthesis of inflammation biomarkers by LPS-stimulated RAW264.7 cells. The inhibitory effects of EDCs can be partially reversed by the addition of an estrogen receptor antagonist for E2 and BPA, and an androgenic receptor antagonist for <em>DHT</em>. The inhibition of inflammatory response in stimulated RAW264.7 cells may be a useful bioassay model for monitoring estrogenic and androgenic pollutants.

Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert <em>5α</em>-androstane-3α,17β-diol (DIOL) to dihydrotestosterone (<em>DHT</em>). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to <em>DHT</em>. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the <em>5α</em>-reductase inhibitor, dutasteride, decreased <em>DHT</em> levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of <em>DHT</em> synthesis provides a therapeutic strategy to inhibit CRPC development and growth.

Androgenic compounds have been implicated in induction of endometrial atrophy yet the mechanisms of androgen effects on human endometrium have not been well studied. We hypothesized that androgens may promote their endometrial effects via modulation of progesterone receptor (PR) expression.

Proliferative phase endometrial samples were collected at the time of hysterectomy. We evaluated the effect of the potent androgen <em>5α</em>-dihydrotestosterone (<em>DHT</em>) on endometrial PR expression by treating human endometrial explants, endometrial stromal cells, and Ishikawa cells with <em>DHT</em>. Ishikawa cells were also treated with <em>DHT</em> ± the androgen receptor (AR) blocker flutamide. The PR-B, total PR messenger RNA (mRNA), and PR protein expression were assessed. Expression of cyclin D1 and D2 was checked as markers of cell proliferation.

As expected, estradiol induced PR expression in isolated stromal cells, endometrial epithelial cells, and tissue explants. The DHT treatment also resulted in increased PR expression in endometrial explants and Ishikawa cells but not in stromal cells. Further, protein levels of both nuclear PR isoforms (PR-A and PR-B) were induced with the DHT treatment. Although flutamide treatment alone did not affect PR expression, flutamide diminished androgen-induced upregulation of PR in both endometrial explants and Ishikawa cells. Although estradiol induced both cyclin D1 and cyclin D2 mRNA, DHT did not induce these markers of cell proliferation.

Androgens may mediate endometrial effects through upregulation of PR gene and protein expression. Endometrial PR upregulation by androgens is mediated, at least in part, through AR.

The herbicide linuron (LIN) is an endocrine disruptor with an anti-androgenic mode of action. The objectives of this study were to (1) improve knowledge of androgen and anti-androgen signaling in the teleostean ovary and to (2) assess the ability of gene networks and machine learning to classify LIN as an anti-androgen using transcriptomic data. Ovarian explants from vitellogenic fathead minnows (FHMs) were exposed to three concentrations of either <em>5α</em>-dihydrotestosterone (<em>DHT</em>), flutamide (FLUT), or LIN for 12h. Ovaries exposed to <em>DHT</em> showed a significant increase in 17β-estradiol (E2) production while FLUT and LIN had no effect on E2. To improve understanding of androgen receptor signaling in the ovary, a reciprocal gene expression network was constructed for <em>DHT</em> and FLUT using pathway analysis and these data suggested that steroid metabolism, translation, and DNA replication are processes regulated through AR signaling in the ovary. Sub-network enrichment analysis revealed that FLUT and LIN shared more regulated gene networks in common compared to <em>DHT</em>. Using transcriptomic datasets from different fish species, machine learning algorithms classified LIN successfully with other anti-androgens. This study advances knowledge regarding molecular signaling cascades in the ovary that are responsive to androgens and anti-androgens and provides proof of concept that gene network analysis and machine learning can classify priority chemicals using experimental transcriptomic data collected from different fish species.